These studies continue to be concerned mainly with two biotin enzymes, oxalacetate-methylmalonyl-CoA transcarboxylase from Propionibacterium and pyruvate carboxylase from several sources including yeast, Pseudomonas, chicken liver and cultured cells of murine origin. For the transcarboxylase, studies are in progress to try to crystallize the large form of the native enzyme (26S) and the central subunit (12S). If successful, X-ray and EM studies of the crystals will be performed. The central 12S subunit is a hexamer and its dissociative and associative properties will be examined. Transcarboxylases from various species of Propionibacterium will be prepared and compared for structural and other properties. Pyruvate carboxylase from Pseudomonas will be studied to elucidate the structural and functional roles of the two polypeptides which comprise this enzyme. Photo-affinity labeled analogs of acetyl-CoA and ATP will be used to study the regulatory and catalYtic sites of several species of pyruvate carboxylase. The pre-adipocyte murine cell line, 3T3-L1, will be used to determine the rates of synthesis and degradation of the apoenzyme and holoenzyme of pyruvate carboxylase and the effects of insulin and c-AMP on these processes.